Hemoglobin Electrophoresis

Electrophoresis
Electrophoresis is a means of separating hemoglobin's.
It depends on the migration of the hemoglobin
molecules dissolved in a buffer on, or in, a supporting
medium when an electric current is passed through
them.
Hemoglobin electrophoresis
Is a test that measures the different types of the
oxygen-carrying substance (hemoglobin) in the blood.
Hemoglobin electrophoresis is performed to find out
abnormal forms of hemoglobin (hemoglobinopathy).
Hemoglobin Electrophoresis
Many different types of hemoglobin (Hb) exist.
The most common ones are HbA, HbA2, HbF,
HbS, HbC, Hb H, and Hb M.
Healthy adults only have significant levels of
HbA and HbA2.
Some people may also have small amounts of
HbF (which is the main type of hemoglobin in an
unborn baby's body). Certain diseases are
associated with high HbF levels (when HbF is
more than 2% of the total hemoglobin).
Hemoglobin Electrophoresis
Hb S is an abnormal form of hemoglobin
associated with sickle cell anemia. In people
with this condition, the red blood cells have a
crescent or sickle shape. These misformed cells
then break down, or can block small blood
vessels.
Hb C is an abnormal form of hemoglobin
associated with hemolytic anemia. The
symptoms are much milder than they are in
sickle cell anemia.
Normal Values
In adults:
• Hgb A1 : 95% to 98%
• Hgb A2 : 2% to 3%
• Hgb F : 0.8% to 2%
• Hgb S : 0%
• Hgb C : 0%
In infants and children:
• Hgb F (newborn) : 50% to 80%
• Hgb F (6 months) : 8%
• Hgb F (over 6 months) : 1% to 2%
Methods of Electrophoresis

1-Cellulose Acetate At Alkaline pH

2- Citrate Agar Electrophoresis ( acid pH)

1-Cellulose Acetate At Alkaline pH
Cellulose acetate Hb electrophoresis at
alkaline pH is the primary screening
procedure used to detect variant (abnormal)
Hbs, of which there are several hundreds.
The major portion of normal adult Hb is A.
In addition, up to 3.5% Hb A2 is normally
present, along with less than 2% Hb F. The
more common mutant Hbs are S, C, E, D,
G, and lepore.
1-Cellulose Acetate At Alkaline pH
When an abnormal Hb is detected on
cellulose acetate electrophoresis at an alkaline
pH (8.2-8.6) further testing is frequently
indicated: test for Hb S, quantitation of Hb A2
and F, and citrate agar gel; acid/alkaline
globin chain or neutral pH electrophoresis
may also be warranted.
Principle of Cellulose Acetate
In an alkaline pH (8.2-8.6)
Hb is a negatively charged
molecule and will migrate
toward the anode (+). The
various Hbs moves at
different rates depending
on their net negative
charge, which in turn is
controlled by the
composition (amino acids)
of the Hb molecule (globin
chain).
Principle of Cellulose Acetate
The red cell hemolysate (red blood cell
membranes are destroyed to free the Hb
molecules for testing) is placed in a cellulose
acetate membrane, which is positioned in an
electrophoresis tray with the inoculated
hemolysate near the cathode (-).
Principle of Cellulose Acetate
One end of the cellulose acetate strip is immersed
in the buffer (pH 8.2-8.6) on the cathode side and
the other end is placed in the buffer on the anode
(+) side. An electric current of specific voltage is
allowed to run for a timed period.
During electrophoresis, the Hb molecules migrate
toward the anode because of their negative charge.
The difference in the net charge of the Hb
molecule determines its mobility and manifests its
self by the speed with which it migrates to the
positive pole.
Principle of cellulose acetate
The cellulose acetate membrane is then stained in
order to color the proteins (Hbs). By noting the
distance each Hb has migrated and comparing
this distance with the migration distance of
known controls, the types of hemoglobins may be
identified.

Example of the fast Hbs are Hb Bart’s and the

two fastest variants Hb H and I, while Hb C is the
slowest common Hb.
 A/SDG

A/SDG

SDG/ACEO

SDG/ACEO

A/F

A/A2CEO
2- Citrate Agar Electrophoresis ( acid pH)

Citrate agar separates Hb fractions that

migrate together on cellulose acetate agar.

All Hb specimens that show an abnormal

electrophoretic pattern in alkaline media
(cellulose acetate agar) should undergo
electrophoresis on an acid citrate agar.
2- Citrate Agar Electrophoresis ( acid pH)
Citrate agar electrophoresis is used to confirm variant
Hbs and further differentiates Hb S from Hb D and G,
and Hb C from Hb E, O Arab, and CHarlem. .

The procedure should not be used as a screening

procedure because many abnormal Hbs migrate with
Hb A. However, this procedure is the method of
choice when examining newborns (cord blood
specimens) and infants under 3 months of age for
some abnormal Hbs such as S and C because the test
is able to detect quantities of Hb not easily seen by
other techniques.
C s A F
D
G
A2
C S A F
D
E
G
A
2